From CLARITY Wiki
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==Imaging techniques== |
==Imaging techniques== |
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| + | Imaging should be performed using a microscopy technique that focuses on one plane within the sample at a time. Standard light microscopy will not be suitable for the optically transparent, intact tissue samples as light will penetrate the entire sample and fluorescence will be emitted from everywhere. Thus, the signal from the focused plane will be diluted and probably lost by the signal coming from everywhere else in the sample, causing the sample to look like a blob of autofluorescence under the light microscope. |
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===Confocal microscopy=== |
===Confocal microscopy=== |
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Revision as of 19:16, 20 April 2014
Following sample clearing in the clearing solution, the hydrogel-embedded tissue is washed in PBST buffer to remove SDS micelles. If the sample was labelled with fluorescent proteins prior to CLARITY processing (from a transgenic mouse line, for example), the proteins will be attached to the hydrogel matrix, and the sample can be imaged immediately after the PBST washing step. Otherwise, the clarified samples can be labelled via immunostaining and then prepared for imaging.
Contents |
Sample preparation
Refractive index matching solutions
FocusClear
Sample mounting
Imaging techniques
Imaging should be performed using a microscopy technique that focuses on one plane within the sample at a time. Standard light microscopy will not be suitable for the optically transparent, intact tissue samples as light will penetrate the entire sample and fluorescence will be emitted from everywhere. Thus, the signal from the focused plane will be diluted and probably lost by the signal coming from everywhere else in the sample, causing the sample to look like a blob of autofluorescence under the light microscope.
Confocal microscopy
(eventually give own page)
Advantages
Disadvantages
Light sheet microscopy
(eventually give own page)